Stable binding of human XPC complex to irradiated DNA confers strong discrimination for damaged sites

Nucleotide excision repair (NER) of DNA damage requires an efficient means of discrimination between damaged and non-damaged DNA. Cells from humans wi th xeroderma pigmentosum group C do not perform NER in the bulk of the geno me and are corrected by XPC protein, which forms a complex with hHR23B prot ein. This complex preferentially binds to some types of damaged DNA, but th e extent of discrimination in comparison to other NER proteins has not been clear. Recombinant XPC, hHR23B, and XPC-hHR23B complex were purified. In a reconstituted repair system, hHR23B stimulated XPC activity tenfold. Elect rophoretic mobility-shift competition measurements revealed a 400-fold pref erence for binding of XPC-hHR23B to UV damaged over non-damaged DNA. This d amage preference is much greater than displayed by the XPA protein. The dis crimination power is similar to that determined here in parallel for the XP -E factor UV-DDB, despite the considerably greater molar affinity of UV-DDB for DNA. Binding of XPC-hHR23B to UV damaged DNA was very fast. Damaged DN A-XPC-hHR23B complexes were stable, with half of the complexes remaining fo ur hours after challenge with excess W-damaged DNA at 30 degrees C. XPC-hHR 23B had a higher level of affinity for (6-4) photoproducts than cyclobutane pyrimidine dimers, and some affinity for DNA treated with cisplatin and al kylating agents. XPC-hHR23B could bind to single-stranded M13 DNA, but only poorly to single-stranded homopolymers. The strong preference of XPC compl ex for structures in damaged duplex DNA indicates its importance as a prima ry damage recognition factor in non-transcribed DNA during human NER. (C) 2 000 Academic Press.